This invention relates to the treatment of neoplastic disease and other pathological conditions characterized by cellular hyperproliferation and loss of regulated growth and motility. More specifically, this invention provides methods for the identification and characterization of agents which modulate PTEN, PI-3 kinase and AKT activity.
Various scientific and scholarly articles are cited throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The reversible phosphorylation of proteins and lipids is critical to the control of signal transduction in mammalian cells and is regulated by kinases and phosphatases (Hunter 1995). The product of the tumor suppressor gene PTEN/MMAC (hereafter termed PTEN) was identified as a dual specificity phosphatase and has been shown to dephosphorylate inositol phospholipids in vivo (Li et al Science 1997, Steck et al 1997, Li et al Cancer Res 1997,Myers et al, 1997, Myers et al 1998, Maehama et al, 1998, Stambolic et al 1998, Wu et al 1998). The PTEN gene, which is located on the short arm of chromosome 10 (10q23), is mutated in 40-50% of high grade gliomas as well as many other tumor types, including those of the prostate, endometrium, breast, and lung (Li et al, Science 1997, Steck et al 1997, Maier et al 1998). In addition, PTEN is mutated in several rare autosomal dominant cancer predisposition syndromes, including Cowden disease, Lhermitte-Duclos disease and Bannayan-Zonana syndrome (Liaw et al 1997, Myers et al AJHG 1997, Maehama et al TCB 1999, Cantley and Neel 1999). Furthermore, the phenotype of PTEN-knockout mice revealed a requirement for this phosphatase in normal development and confirmed its role as a tumor suppressor (Podsypanina et al PNAS 1999, Suzuki et al Curr Biol 1998, Di Christofano et al Nat Gen 1998).
PTEN is a 55 kDa protein comprising an N-terminal catalytic domain, identified as a segment with homology to the cytoskeletal protein tensin and containing the sequence HC(X)5R (SEQ ID NO: 22), which is the signature motif of members of the protein tyrosine phosphatase family, and a C-terminal C2 domain with lipid-binding and membrane-targeting functions (Lee et al Cell 1999). The sequence at the extreme C-terminus of PTEN is similar to sequences known to have binding affinity for PDZ domain-containing proteins. PTEN is a dual specificity phosphatase that displays a pronounced preference for acidic substrates (Myers et al PNAS 1997). Importantly, PTEN possesses lipid phosphatase activity, preferentially dephosphorylating phosphoinositides at the D3 position of the inositol ring. It is one of two enzymes known to dephosphorylate the D3 position in inositol phospholipids.
Since solid tumor progression is dependent on the induction of angiogenic signals and augmented angiogenesis contributes to the high mortality associated with many cancers, there is a need to elucidate the cellular components that participate in these processes. The urgency of such investigations is underscored by the fatal nature of highly malignant brain tumors and the fact that the degree of tumor invasiveness is directly correlated with enhanced angiogenesis. Furthermore, elucidation of cellular components that contribute to the angiogenic switch facilitates the identification of therapeutic agents and delivery methods useful for the treatment of such malignant diseases.
PTEN phosphatase activity has also been implicated in many cellular biochemical reactions. It is an object of the invention to also provide methods for the identification of agents which impact PTEN modulation of immunoreceptors, AKT, PI3 kinase and p53 signaling. Methods of use of agents so identified are also within the scope of the invention.
PTEN is a pivotal signaling molecule which modulates a wide variety of cellular processes. These cellular processes include angiogenesis, cellular migration, immunoreceptor modulation, p53 signaling and apoptotic cell death, PI3 and AKT signaling. Mutations in PTEN have been associated with the highly malignant progression of brain tumors. A hallmark of this malignant progression is a dramatic increase in angiogenesis and invasiveness mediated by the concomitant formation of new blood vessels.
Thus, in accordance with the present invention methods for the treatment of cancer associated with PTEN mutation are provided. Exemplary methods include delivery of a native PTEN encoding nucleic acid to cancer cells such that the native PTEN protein is expressed. Additional methods for the treatment of cancer in accordance with the present invention entail the administration of at least one agent selected from the group consisting of PTEN agonists, PI3 kinase inhibitors and AKT inhibitors. The aforementioned treatment protocols may also comprise the administration of conventional chemotherapeutic agents.
In another aspect of the invention, methods for the prevention of aberrant angiogenesis are also provided. Aberrant angiogenesis is associated with several diseases. These include not only cancer, but autoimmune disease, arthritis, systemic lupus erthymatosis, inflammatory bowel disease, coronary artery disease, cerebrovascular disease, and atherosclerosis. Methods for the administration of at least one agent selected from the group consisting of native PTEN encoding nucleic acids, PTEN agonists, PI3 kinase inhibitors and AKT inhibitors for the inhibition or prevention of aberrant angiogenesis are also disclosed herein.
PTEN has also been implicated in immunoreceptor modulation. Thus, in yet another aspect of the invention, methods for inhibiting the immune response in target cells are provided. PTEN agonists, PI3 kinase inhibitors and/or AKT inhibitors are administered to patients to prevent or inhibit immunoreceptor signaling. Such agents should have efficacy in the treatment of graft rejection or graft versus host disease.
In yet another aspect of the invention, methods for regulating p53 mediated gene expression are also provided. Such methods entail the administration of native PTEN, PTEN agonists and/or PI3 kinase inhibitors or AKT inhibitors to induce functional p53 in tumor cells. Such agents effectively increase chemosensitity and/or radiosensitivity of tumor cells by stimulating p53 mediated apoptotic cell death.
Given the widespread effects of PTEN, methods for identifying agents which modulate PTEN activity are also provided. Exemplary assays include those which assess alterations in activated AKT levels, alterations in microvessel formation, alterations in TSP1 levels, alterations in VEGF levels, alterations in TIMP3 levels, alterations in MMP9 activation and alterations PTEN phosphatase activity levels in the presence and absence of such test agents.
Also provided in accordance with the present invention are high throughput screening methods for identifying small molecules which have affinity for PTEN or fragments thereof. Small molecules so identified are within the scope of the present invention and may optionally be further characterized in the functional assays described above.